The present invention provides improved devices for detecting the presence of an analyte in a sample. Common optical biological sensor devices use fluorescent labels (throughout this text the term xe2x80x9clabeled analoguexe2x80x9d refers to an analogue labeled with a fluorescent label) to signal the presence of analyte.
A well know problem associated with fluorescent dyes and optical measurements is that the fluorescent response (intensity) of an optical sensor is dependent on the intensity of the light that irradiates it. The intensity of the light that strikes the optical sensor is in turn dependent upon the optical path length and the absorptivity and scattering of the media that the light must travel through before it reaches the monitoring device, and the path the fluorescent signal must take in order to reach the detector. In addition, any variation in the intensity of the power output of the light emitting device will be interpreted as a change in the concentration of the analyte.
Various approaches have been used to solve this problem. One strategy is to incorporate a separate internal reference dye in the sensor.
The reference dye can be an organic dye, which fluoresces at a substantially different wavelength than the labeled analogue. The excitation wavelength of the reference dye can be the same as the fluorescent label attached to the analogue or different.
By physically moving the light source and photodetector the intensity of the reference dye can be monitored and optimized. Since the reference dye is in close proximity to the labeled analogue, light emitted from the reference dye and labeled analogue travel substantially the same path to the detector, resulting in a similar attenuation due to scattering, absorption and path length. Therefore, by ratioing the labeled analogue intensity to the reference intensity, any effects due to scattering, absorptivity or path length is removed.
Generally, the fluorescent label and the reference dye absorb and emit at wavelengths different for one another. These absorption and emission wavelengths are inherent to a specific dye/label and limit one""s choice of absorption and emission wavelengths to the available dyes. The colors or wavelengths emitted by the dyes tend to bleed together, and if two dyes are required for a device, two lasers of different wavelength are generally required (in order to obtain good spectral separation between the emission wavelengths of the reference and analogue dye). Using two lasers is not only unwieldy, but expensive. These limitations mean that, in practice, it is very difficult to use different dyes in the same device.
Quantum dots have none of these shortcomings. Quantum dots are particles that measure only a few nanometers in diameter. They come in a nearly unlimited palette of colors and can be linked to other molecules (such as bio-molecules, including proteins and polynucleotides, glass, and plastic) to adjust their solubility. The emission wavelength of quantum dots can be tuned by varying the size of the nanoparticles and can be used to make a rainbow of colors with white light or a single-color laser. Furthermore, the quantum dots have better photostability than traditional reference dyes.
By incorporating quantum dots into an analyte sensing device, an internal reference for quantitative analysis of an analyte is provided. Upon irradiation with light at an absorption wavelength, the quantum dot emits at a significantly different wavelength. In addition, quantum dots have a broad absorption band, but a fixed emission wavelength, which depends on the composition of the quantum dot and its size. A broad absorption band means that a wide range of wavelengths (colors) can be used to excite the quantum dot, and yet the nanoparticle still emits at the same emission wavelength.
One benefit of placing a reference in close proximity to the fluorescent label is the ability to maximize the emission intensity available from both the labeled analogue and the reference dye. When the analyte sensing device is implanted in a human, and the excitation source or laser is above the skin, the invariant (analyte independent) nature of the reference emission may be used to focus the excitation beam on the device, and to spatially locate the implant to optimize its signal to noise ratio. Greater precision in focusing the excitation beam, allows for a lower analyte emission requirement; therefore, a smaller device is possible. Accurate focusing allows for a smaller sensor device.
In the analyte sensing devices of the present invention, quantum dots are used as a reference to generate an analyte invariant reference emission intensity. This reference, in conjunction with a labeled analogue, can be used to quantitatively determine the amount of analyte in a sample. When irradiated or excited with electromagnetic radiation at a specific wavelength, or a specific range of wavelengths, the quantum dot can emit at a different wavelength than at which it was excited.